AKR1B10 confers resistance to radiotherapy via FFA/TLR4/NF-κB axis in nasopharyngeal carcinoma

Abstract

Nasopharyngeal carcinoma (NPC) is one kind of human head and neck cancers with high incidence in Southern China, Southeast Asia and North Africa. In spite of great innovations in radiation and chemotherapy treatments, the 5-year survival rate is not satisfactory. One of the main reasons is resistance to radiotherapy which leads to therapy failure and recurrence of NPC. The mechanism underlying remains to be fully elucidated. Aldo-keto reductase B10 (AKR1B10) plays a role in the formation and development of carcinomas. However, its role in resistance to radiotherapy of NPC is not clear. In this research, the relationships between AKR1B10 expression and the treatment effect of NPC patients, NPC cell survival, cell apoptosis, and DNA damage repair, as well as the effect and mechanism of AKR1B10 expression on NPC radioresistance were explored. A total of 58 paraffin tissues of NPC patients received radiotherapy were collected including 30 patients with radiosensitivity and 28 patients with radioresistance. The relationships between AKR1B10 expression and the treatment effect as well as clinical characteristics were analyzed by immuno-histochemical experiments, and the roles of AKR1B10 in cell survival, apoptosis and DNA damage repair were detected using the AKR1B10 overexpressed cell models. Furthermore the mechanism of AKR1B10 in NPC radioresistance was explored. Finally, the radioresistance effect of AKR1B10 expression was evaluated by the tumor xenograft model of nude mice and the method of radiotherapy. The results showed AKR1B10 expression level was correlated with radiotherapy resistance, and AKR1B10 overexpression promoted proliferation of NPC cells, reduced apoptosis and decreased cellular DNA damage after radiotherapy. The probable molecular mechanism is that AKR1B10 expression activated FFA/TLR4/NF-κB axis in NPC cells. This was validated by using the TLR4 inhibitor TAK242 to treat NPC cells with AKR1B10 expression, which reduced the phosphorylation of NF-κB. This study suggests that AKR1B10 can induce radiotherapy resistance and promote cell survival via FFA/TLR4/NF-κB axis in NPC, which may provide a novel target to fight against radiotherapy resistance of NPC.

Keywords: AKR1B10; FFA/TLR4/NF-κB axis; nasopharyngeal carcinoma; radiotherapy resistance.

Figure 1

The expression of AKR1B10 in patients of radiotherapy resistance and radiotherapy sensitivity. (A) AKR1B10 expression detected by immunochemistry, and images a-d are representative images of 4 levels of expression from 0 to 3 score, such as 0 for no staining (a), 1 for week staining (b), 2 for moderate staining (c), 3 for strong staining (d). (B) Immunohistochemical detection of AKR1B10 expression in radio-sensitive and radio-resistant patients.

Figure 1

The expression of AKR1B10 in patients of radiotherapy resistance and radiotherapy sensitivity. (A) AKR1B10 expression detected by immunochemistry, and images a-d are representative images of 4 levels of expression from 0 to 3 score, such as 0 for no staining (a), 1 for week staining (b), 2 for moderate staining (c), 3 for strong staining (d). (B) Immunohistochemical detection of AKR1B10 expression in radio-sensitive and radio-resistant patients.

Figure 2

AKR1B10 forcing expression promoted proliferation and reduced apoptosis of NPC cells. (A and B) AKR1B10 expression confirmed at CNE-2 cells by Western blot and qRT-PCR. (C and D) The proliferation of CNE-2/AKR1B10 cells verified by clone formation assay. The experiments repeated three times. (E and F) The effect of AKR1B10 expression on CNE-2 cell apoptosis detected by Flow cytometry after IR treatment. (G) Expression levels of cleaved-caspase-3, cleaved-PARP in CNE-2/AKR1B10 cells after IR (6 Gy) 48 hours determinated by Western blot. (H) Quantification and statistics for the cleaved PARP and cleaved Caspase 3 expression. IR: irradiation; Gy: GrayA; AKR1B10; V: vector, psin-EF2-puromycine; CNE-2/A: CNE-2/AKR1B10, AKR1B10 expressed CNE-2 cells; CNE-2/V: CNE-2/AKR1B10 vector, CNE-2 control cells; *p< 0.05.

Figure 3

Evaluation of DNA damage after irradiation in AKR1B10-overexpression NPC cells. (A) The γH2AX staining of CNE-2/AKR1B10 cells (the scale bar represents 50 µm). (B and C) Statistics of the percentage of γH2AX foci and foci greater than 20 in CNE-2/AKR1B10 and CNE-2/vector cells. (D and E) The comet assay of CNE-2/AKR1B10 cells after IR treatment. D is one of the representative Images and E is the statistics result of three expreiments (counting 100 cells per group, Scale bar 200μm). (F) The effect of AKRB10 expression on cell cycle checkpoint proteins expression by Western blot after IR treatment. (G and H) The relative quantification analysis of the change of phosphorylated CHK1 and CHK2 between the control group and the experimental groups. The signals of protein bands relative to β-actin in three experiments were quantified by ImageJ software. IR: irradiation; Gy: GrayA; A: AKR1B10; V: vector, psin-EF2-puromycine; CNE-2/A: CNE-2/AKR1B10, AKR1B10-expressed CNE-2 cells; CNE-2/V: CNE-2/AKR1B10 vector, CNE-2 control cells; p-CHK1: phosphorylated CHK1; p-CHK2: phosphorylated CHK2. *P < 0.05; **P < 0.01.

Figure 4

AKR1B10 activates the NF-κB pathway. (A) The levels of IKBα/NF-κB signaling pathway protein at CNE-2/AKR1B10 cells were assessed by Western blot after IR treatment (0 and 2 Gy) at 4, 12 and 24 h respectively. (B and C) The quantificated statistics results of phosphorylated NF-κB and phosphorylated IKBα protein expression relative to β-actin protein level. The experiments were repeated three times. (D) Construction of AKR1B10-knockdown cell line. (E and F) Knockdown of AKR1B10 downregulates the phosphorylation level of NF-κB. IR: irradiation; Gy: GrayA; A: AKR1B10; V: vector, psin-EF2-puromycine; CNE-2/A: CNE-2/AKR1B10, AKR1B10 expressed CNE-2 cells; CNE-2/V: CNE-2/AKR1B10 vector, CNE-2 control cells; *P < 0.05; **P < 0.01.

Figure 5

Enhanced AKR1B10 expression activates the TLR4/ NF-κB signaling pathway. (A and B) The effect of AKR1B10 expression on FFA content. AKR1B10 expression increases FFA content of CNE-2/AKR1B10 cells (CNE-2/A), meanwhile, knockdown of AKR1B10 expression decreases FFA content. (C) 0, 50, 100 and 200 µM FFA were used to treat CNE-2/AKR1B10 cells and the expression levels of TLR4, p-IKBɑ and p-NF-κB p65 as well as AKR1B10 were detected by western blot. (D) The expression levels of TLR4, p-IKBɑ and p-NF-κB p65 as well as AKR1B10 were further confirmed in the CNE-2/vector and CN-2/AKR1B10 cells after treated with 100 μM FFA. (E) The effect of FFA on NF-κB p65 entry into the nucleus. (F) The effect of FFA inhibitors on TLR4 signaling pathway. A: AKR1B10; V: vector, psin-EF2-puromycin; CNE-2/A: CNE-2/AKR1B10, AKR1B10 expressed CNE-2 cells; CNE-2/V: CNE-2/AKR1B10 vector, CNE-2 control cells; FFA: Free fatty acid; H3: H3 histone; TLR4-IN-C34:TLR4 inhibitor; p-IKBɑ: phosphorylated IKBα; p-NF-κB: phosphorylated NF-κB. *P < 0.05.

Figure 6

AKR1B10 promotes NPC radioresistance in vivo. (A) Whole tumors excised from mice injected with CNE-2/Vector, CNE2/Vector+IR, CNE2/AKR1B10, CNE2/AKR1B10+IR. (B and C) Tumor volumes and weight were determined. (D) Analysis of tumor tissue by HE staining and immunohistochemical staining. IR: irradiation; CNE-2/A: CNE-2/AKR1B10, AKR1B10-expressed CNE-2 cells; CNE-2/V: CNE-2/AKR1B10 vector, CNE-2 control cells;*P < 0.05; **P < 0.01.

Figure 7

Proposed Schematic for AKR1B10 activating TLR4/NF-κB signaling pathway by promoting FFA synthesis in NPC. AKR1B10-induced FFA synthesis activates the TLR4/ NF-κB signaling axis, IKBα phosphorylation and NF-κB nuclear translocation, which then regulates cell cycle arrest and DNA damage repair. IR: irradiation; FFA: Free fatty acid; →, activation or upregulation; ┴ inhibition or downregulation.